Placental tissue grafts produced by chemical dehydration/freeze-drying and methods for making and using the same

ABSTRACT

Described herein are placental tissue grafts produced by chemical dehydration followed by freeze-drying the placental tissue to produce the tissue graft. The tissue grafts retain their biological properties preferably at the same level as the placental tissues before they are processed. The placental tissue grafts have numerous medical applications. Methods for making the tissue graft compositions are also described herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/691,508, filed Nov. 30, 2012, which claims the benefit of U.S.Provisional Application No. 61/566,057, filed Dec. 2, 2011, each ofwhich are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to placental tissue grafts, methods of preparingthe tissue grafts, compositions and uses thereof.

SUMMARY OF THE INVENTION

Described herein are placental tissue grafts produced by chemicaldehydration followed by freeze-drying the placental tissue to producethe tissue graft. The tissue grafts preferably are undamaged during thedehydration process and retain biological properties preferably at thesame level as the placental tissues before they are processed. Theplacental tissue grafts have numerous medical applications. Methods formaking the tissue graft compositions are also described herein.

The advantages of the invention will be set forth in part in thedescription which follows, and in part will be apparent from thedescription, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 is an overview flow chart of the process for making the tissuegraft compositions described herein.

DETAILED DESCRIPTION

Before the present articles and methods are disclosed and described, itis to be understood that the aspects described below are not limited tospecific articles, compositions, preparations and/or methods, or uses assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a bioactive agent” includes mixtures of two or more suchagents, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally cleaning step” means thatthe cleaning step may or may not be performed.

The term “subject” as used herein is any vertebrate organism, includingmammals. In one embodiment, the subject is a human.

The term “placenta” or “harvested placenta” as used herein refers to theplacenta obtained from a female mammal during or after a child birth asis well known in the art.

The term “placental tissue” refers to one or more of the individualcomponents of the placenta (but not the entire placenta). Suchcomponents are well known in the art and include, amnion, chorion,Wharton's jelly and any combination thereof. Included within the term“amnion” are unmodified and modified amnion. Modified amnion includesamnion in which the epithelial layer has been removed (mechanically,chemically or enzymatically) while retaining the fibroblast cellularlayer, amnion which has been completely decellularized as well as amnionwhich retains the epithelial layer while having the fibroblast layerremoved.

The term “placental tissue graft” refers to one or more layers ofplacental tissue which have been processed as per this invention and aresuitable for use as a graft in treating a condition in a mammal such asa human.

Titles or subtitles may be used in the specification for the convenienceof a reader, which are not intended to influence the scope of thepresent invention. Additionally, some terms used in this specificationare more specifically defined below.

I. Tissue Grafts and Methods for Making Thereof

Described herein are tissue grafts and methods of making and usingthereof. In one aspect, the tissue grafts are produced by a processcomprising:

a. chemically dehydrating a harvested placental tissue; and

b. freeze-drying the placental tissue to produce the tissue graft.

In some embodiments, the placental tissue graft comprises at least twolayers of chorion, at least two layers of amnion membrane, or at leastone layer of chorion and at least one layer of amnion membrane.

In some embodiments, the placental tissue graft comprises amnionmembrane/Wharton's jelly/chorion. In some embodiments, the placentaltissue graft comprises at least one layer of chorion and at least onelayer of amnion membrane, and further comprises Wharton's jellyintermediate between the amnion membrane and chorion membrane.

In another aspect, provided is a method for producing a placental tissuegraft, which method comprises:

a. chemically dehydrating a placental tissue; and

b. freeze-drying the placental tissue to produce the tissue graft.

In some embodiments, the method further comprises, prior to step (a),laminating two or more layers selected from amnion, Wharton's jellyand/or chorion membranes prior to dehydration. In some embodiments, twoor more layers of amnion membranes are laminated. In some embodiments,two or more layers of chorion membranes are laminated. In someembodiments, at least one layer of amnion membrane and at least onelayer of chorion membrane are laminated. In some embodiments, at leastone layer of amnion membrane, at least one layer of Wharton's jelly andat least one layer of chorion membrane are laminated.

In some embodiments, the method further comprises, prior to step (a),chemically decontaminating the placental tissue. In some embodiments,the decontaminating step is prior to the laminating step. In someembodiments, the placental tissue is decontaminated by being soaked inan antibiotic solution and/or a detergent.

In some embodiments, the method further comprises physically cleaningthe placental tissue to remove blood clots and other contaminates.

In some embodiments, step (a) comprises contacting the placental tissuewith a sufficiently amount of a polar organic solvent for a sufficienttime in order to substantially or completely remove residual waterpresent in the placental tissue. In some embodiments, step (a) comprisessoaking the placental tissue in a polar organic solvent at roomtemperature. In some embodiments, the polar organic solvent comprises analcohol, a ketone, an ether, an aldehyde, or any combination thereof. Insome embodiments, the polar organic solvent comprises DMSO, acetone,tetrahydrofuran, ethanol, isopropanol, or any combination thereof.

In some embodiments, the placental tissue is freeze-dried at atemperature of from −50° C. to −80° C.

In some embodiments, step (b) comprises (1) laying one or morechemically dehydrated placental tissue on a substrate and (2) insertingthe substrate with the one or more placental tissue in a freeze-dryer.In some embodiments, the substrate is a bowl, pan, screen, frame, ordrying fixture.

The method and additional steps that can be included in the method areexemplified in detail below.

FIG. 1 depicts an exemplary, non-limiting overview (100) and certainaspects of the steps to harvest, process, and prepare placental tissuesfor use in the preparation of the tissue grafts described herein. Moredetailed descriptions and discussion regarding each individual step willfollow. Initially, the placenta is collected (step 110). The material ispreserved and transported in conventional tissue preservation manner toa suitable processing location or facility for check-in and evaluation(step 120). Gross processing, handling, and separation of the placentalor placental tissue then takes place (step 130). Acceptable placenta orplacental tissue is then decontaminated (step 140). Afterdecontamination, the placental tissue is chemically dehydrated andfreeze-dried (step 145). Each step is described in detail below.

Initial Tissue Collection (Step 110)

The components used to produce the tissue grafts are derived from theplacenta. The source of the placenta can vary. In one aspect, theplacenta is derived from a mammal such as human and other animalsincluding, but not limited to, cows, pigs, and the like. In the case ofhumans, the recovery of the placenta originates in a hospital, where itis collected during a Cesarean section birth. The donor, referring tothe mother who is about to give birth, voluntarily submits to acomprehensive screening process designed to provide the safest tissuepossible for transplantation. The screening process preferably tests forantibodies to the human immunodeficiency virus type 1 and type 2(anti-HIV-1 and anti-HIV-2), antibodies to the hepatitis B virus(anti-HBV) hepatitis B surface antigens (HBsAg), antibodies to thehepatitis C virus (anti-HCV), antibodies to the human T-lymphotrophicvirus type I and type II (anti-HTLV-I, anti-HTLV-II), CMV, and syphilis,and nucleic acid testing for human immune-deficiency virus type 1(HIV-1) and for the hepatitis C virus (HCV), using conventionalserological tests. The above list of tests is exemplary only, as more,fewer, or different tests may be desired or necessary over time or basedupon the intended use of the grafts, as will be appreciated by thoseskilled in the art.

Based upon a review of the donor's information and screening testresults, the donor will either be deemed acceptable or not. In addition,at the time of delivery, cultures are taken to determine the presence ofbacteria, for example, Clostridium or Streptococcus. If the donor'sinformation, screening tests, and the delivery cultures are allsatisfactory (i.e., do not indicate any risks or indicate acceptablelevel of risk), the donor is approved by a medical director and thetissue specimen is designated as initially eligible for furtherprocessing and evaluation.

Human placentas that meet the above selection criteria are preferablybagged in a saline solution in a sterile shipment bag and stored at alow temperature (e.g., −1° C.-10° C., 0° C.-10° C., 1° C.-10° C., or 0°C.-5° C.), such as in a container of wet ice, for shipment to aprocessing location or laboratory for further processing.

If the placenta is collected prior to the completion or obtaining ofresults from the screening tests and delivery cultures, such placenta orplacental tissue is labeled and kept in quarantine. The placenta isapproved for further processing only after the required screeningassessments and delivery cultures, which declare the placenta orplacental tissue safe for handling and use, are satisfied and obtainfinal approval from a medical director.

Material Check-in and Evaluation (Step 120)

Upon arrival at the processing center or laboratory, the shipment isopened and verified that the sterile shipment bag/container is stillsealed and in the coolant, that the appropriate donor paperwork ispresent, and that the donor number on the paperwork matches the numberon the sterile shipment bag containing the placenta or placenta tissue.The sterile shipment bag containing the tissue is then stored at a lowtemperature (e.g., −1° C.-10° C., 0° C.-10° C., 1° C.-10° C., or 0°C.-5° C.), such as in a refrigerator until ready for further processing.

Gross Tissue Processing (Step 130)

When the placenta or placental tissue is ready to be processed further,the sterile supplies necessary for processing the placenta or placentaltissue further are assembled in a staging area in a controlledenvironment and are prepared for introduction into a controlledenvironment. In one aspect, the placenta is processed at roomtemperature. If the controlled environment is a manufacturing hood, thesterile supplies are opened and placed into the hood using conventionalsterilization techniques. If the controlled environment is a clean room,the sterile supplies are opened and placed on a cart covered by asterile drape. The work surfaces are covered by sterile drape usingconventional sterilization techniques, and the sterile supplies and theprocessing equipment are placed onto the sterile drape, again usingconventional sterilization techniques.

Processing equipment is decontaminated according to conventional andindustry-approved decontamination procedures and then introduced intothe controlled environment. The equipment is strategically placed withinthe controlled environment to minimize the chance for the equipment tocome in proximity to or is inadvertently contaminated by the tissuespecimen.

Next, the placenta is removed from the sterile shipment bag andtransferred aseptically to a sterile processing basin within thecontrolled environment. The sterile basin contains hyperisotonic salinesolution (e.g., 18% NaCl) that is at room or near room temperature. Theplacenta is gently massaged to help separate blood clots and to allowthe placental tissue to reach room temperature, which facilitates theseparation of the placental components from each other (e.g., amnionmembrane and chorion). After having warmed up to the ambient temperature(e.g., after about 10-30 minutes), the placenta is then removed from thesterile processing basin and laid flat on a processing tray with theamnion membrane layer facing down for inspection.

The placenta is examined for discoloration, debris or othercontamination, odor, and signs of damage. The size of the placenta orplacental tissue is also noted. A determination is made, at this point,as to whether the placenta or placental tissue is acceptable for furtherprocessing. In one embodiment, the placenta is acceptable for furtherprocessing if no discoloration, debris, unacceptable odor, and/or signsof damage is observed.

In one aspect, if the placenta is deemed acceptable for furtherprocessing, the amnion membrane, Wharton's jelly, and chorion arecollectively dissected from the placenta. In this aspect, the amnionmembrane, Wharton's jelly, and chorion are not separated from one, andthis resulting placental tissue is further processed as discussed below.

In another aspect, the amnion membrane and chorion of the placenta canbe carefully separated. In one aspect, the materials and equipment usedin this procedure include a processing tray, 18% saline solution,sterile 4×4 sponges, and two sterile Nalgene jars. The placenta is thenclosely examined to find an area (typically a corner) in which theamnion membrane can be separated from the chorion. The amnion membraneappears as a thin, opaque layer on the chorion.

The fibroblast layer is identified by gently contacting each side of theamnion membrane with a piece of sterile gauze or a cotton tippedapplicator. The fibroblast layer will stick to the test material. Theamnion membrane is placed into processing tray basement membrane layerdown. Using a blunt instrument, a cell scraper or sterile gauze, anyresidual blood is also removed. This step is done with adequate care,again, so as not to tear the amnion membrane. The cleaning of the amnionmembrane is complete once the amnion membrane is smooth and opaque-whitein appearance.

In certain aspects, the intermediate tissue layer is removed from theamnion membrane. This can be performed by peeling the intermediatetissue layer from the amnion membrane. Alternatively, the intermediatetissue layer can be removed from the amnion membrane by wiping theintermediate tissue layer with a gauze or other suitable wipe. Theresulting amnion can be subsequently decontaminated using the processdescribed below. The intermediate tissue layer does not require anyadditional processing and can be used as-is.

In certain aspects, the Wharton's jelly can optionally be isolated usingthe following procedure. Using a scalpel or scissors, the umbilical cordis dissected away from the chorionic disk. Once the veins and the arteryhave been identified, the cord is dissected lengthwise down one of theveins or the artery. Once the umbilical cord has been dissected,surgical scissors and forceps can be used to dissect the vein and arterywalls from the Wharton's jelly. Next, the outer layer of amnion membraneis removed from the Wharton's jelly by cutting the amnion membrane.After removing the amnion membrane from the Wharton's jelly, theWharton's jelly can be cut into strips. In one aspect, the strips areapproximately 1-4 cm by 10-30 cm with an approximate thickness of 1.25cm; however, other sizes and/or thicknesses are possible depending onapplication.

Chemical Decontamination (Step 140)

Any of the placenta or placental tissues discussed herein can bechemically decontaminated using the techniques described below. In oneaspect, the placenta or placental tissue is decontaminated at roomtemperature. In one aspect, the amnion membrane produced in step 130 canbe placed into a sterile Nalgene jar for the next step. In one aspect,the following procedure can be used to clean the amnion membrane. EachNalgene jar is aseptically filled with 18% saline hypertonic solutionand sealed (or sealed with a top). The jar is then placed on a rockerplatform and agitated for between 30 and 90 minutes, which furthercleans the amnion membrane of contaminants. If the rocker platform isnot in the critical environment (e.g., the manufacturing hood), theNalgene jar is returned to the controlled/sterile environment andopened. Using sterile forceps or by aseptically decanting the contents,the amnion membrane is gently removed from the Nalgene jar containingthe 18% hyperisotonic saline solution and placed into an empty Nalgenejar. This empty Nalgene jar with the amnion membrane is then asepticallyfilled with a pre-mixed antibiotic solution. In one aspect, the premixedantibiotic solution is composed of a cocktail of antibiotics, such asStreptomycin Sulfate and Gentamicin Sulfate. Other antibiotics, such asPolymixin B Sulfate and Bacitracin, or similar antibiotics now availableor available in the future, are also suitable. Additionally, it ispreferred that the antibiotic solution be at room temperature when addedso that it does not change the temperature of or otherwise damage theamnion membrane. This jar or container containing the amnion membraneand antibiotics is then sealed or closed and placed on a rocker platformand agitated for, preferably, between 60 and 90 minutes. Such rocking oragitation of the amnion membrane within the antibiotic solution furthercleans the tissue of contaminants and bacteria. Optionally, the amnionmembrane can be washed with a detergent. In one aspect, the amnionmembrane can be washed with 0.1 to 10%, 0.1 to 5%, 0.1 to 1%, or 0.5%Triton-X wash solution.

If the rocker platform is not in the critical environment (e.g., themanufacturing hood), the jar or container containing the amnion membraneand antibiotics is then returned to the critical/sterile environment andopened. Using sterile forceps, the amnion membrane is gently removedfrom the jar or container and placed in a sterile basin containingsterile water or normal saline (0.9% saline solution). The amnionmembrane is allowed to soak in place in the sterile water/normal salinesolution for at least 10 to 15 minutes. The amnion membrane may beslightly agitated to facilitate removal of the antibiotic solution andany other contaminants from the tissue. After at least 10 to 15 minutes,the amnion membrane is ready to be dehydrated and processed further.

In the case when the chorion is to be used, the following exemplaryprocedure can be used. After separation of the chorion from the amnionmembrane and removal of clotted blood from the fibrous layer, thechorion is rinsed in 18% saline solution for 15 minutes to 60 minutes.During the first rinse cycle, 18% saline is heated in a sterilecontainer using a laboratory heating plate such that the solutiontemperature is approximately 48° C. The solution is decanted, thechorion tissue is placed into the sterile container, and decanted salinesolution is poured into the container. The container is sealed andplaced on a rocker plate and agitated for 15 minutes to 60 minutes.After 1 hour agitation bath, the chorion tissue was removed and placedinto second heated agitation bath for an additional 15 minutes to 60minutes rinse cycle. Optionally, the chorion tissue can be washed with adetergent (e.g., Triton-X wash solution) as discussed above for theamnion membrane. The container is sealed and agitated without heat for15 minutes to 120 minutes. The chorion tissue is next washed withdeionized water (250 milliliter (mL) of DI water×4) with vigorous motionfor each rinse. The tissue is removed and placed into a container of1×PBS w/EDTA solution. The container is sealed and agitated for 1-8hours at controlled temperature. The chorion tissue is removed andrinsed using sterile water. A visual inspection was performed to removeany remaining discolored fibrous blood material from the chorion tissue.The chorion tissue should have a cream white visual appearance with noevidence of brownish discoloration.

In the case of the decontamination of Wharton's jelly, it can betransferred to a sterile Nalgene jar. Next, room temperature 18%hypertonic saline solution is added to rinse the tissue and the jar issealed. The jar is agitated for 30 to 60 minutes. After incubation, thejar is decontaminated and returned to the sterile field. The tissue istransferred to a clean sterile Nalgene jar and prewarmed (about 48° C.)18% NaCl is added. The container is sealed and placed on a rocker plateand agitated for 60 to 90 minutes.

In one aspect, after the rinse, the jar is decontaminated and returnedto the sterile field. The tissue is removed and placed into anantibiotic solution. The container is sealed and agitated for 60 to 90minutes on a rocker platform. Following incubation, the jar may berefrigerated at 1 to 10° C. for up to 24 hours. The Wharton's jelly isnext transferred to a sterile basin containing approximately 200 mL ofsterile water. The tissue is rinsed for 1-2 minutes and transferred to asterile Nalgene jar containing approximately 300 mL of sterile water.The jar is sealed and placed on the rocker for 30 to 60 minutes. Afterthe incubation, the jar is returned to the sterile field. The Wharton'sjelly should have a cream white visual appearance with no evidence ofbrownish discoloration. The tissue is ready for further processing.

In other aspects, when the placental tissue is amnion membrane,Wharton's jelly, and chorion that has not been separated, the placentaltissue can be decontaminated using any of the techniques and solutionsdescribed above (e.g., antibiotic solutions, detergents such as TritonX, etc.).

In certain aspects, it is desirable to remove epithelium layer presenton the amnion membrane after chemical decontamination. In one aspect,the epithelium layer present on the amnion membrane is substantiallyremoved in order to expose the basement layer of the amnion membrane.The term “substantially removed” with respect to the amount ofepithelium removed is defined herein as removing greater than 50%,greater than 60%, greater than 70%, greater than 80%, greater than 90%,greater than 95%, or greater than 99% of the epithelial cells from theamnion. The presence or absence of epithelial cells remaining on theamnion membrane can be evaluated using techniques known in the art. Forexample, after removal of the epithelial cell layer, a representativetissue sample from the processing lot is placed onto a standardmicroscope examination slide. The tissue sample is then stained usingEosin Y Stain and evaluated. The sample is then covered and allowed tostand. Once an adequate amount of time has passed to allow for staining,visual observation is done under magnification.

The epithelium layer can be removed by techniques known in the art. Forexample, the epithelium layer can be scraped off of the amnion using acell scraper. Other techniques include, but are not limited to, freezingthe membrane, physical removal using a cell scraper, or exposing theepithelial cells to nonionic detergents, anionic detergents, andnucleases. The de-epithelialized tissue is then evaluated to determinethat the basement membrane has not been compromised and remains intact.This step is performed after completion of the processing step and thetissue has been dehydrated as described in the next section. Forexample, a representative sample graft is removed for microscopicanalysis. The tissue sample is place onto a standard slide and 100microliter (μL) of Eosin Y stain is applied to the sample and allowed toset. The tissue sample is then examined under magnification. Cellularmaterial will stain dark indicating the presence of cells. If no stainedcells are present, de-epithelization has been achieved.

Dehydration (Step 145)

One or more placental tissues prepared above can be dehydrated toproduce dehydrated placental tissue grafts. In the case when two or moreplacental tissues are used, the tissue is a laminate. In one aspect, twoor more membranes are laminated prior to dehydration. For example, alaminate composed of amnion, amnion membrane, chorion, Wharton's jelly,or any combination thereof can be produced. In one aspect, the tissue isan amnion/chorion laminate. In another aspect, the tissue has at leasttwo layers of chorion, at least two layers of amnion, or at least onelayer of chorion and at least one layer of amnion. In a further aspect,the placental tissue has a plurality chorion and/or amnion membraneslaminated to one another.

Techniques for producing laminated tissue are know in the art. Forexample, a layer of amnion optionally with substantially no epithelialcells on the surface of the basement membrane can be applied to afixture, one or more amnion and/or chorion membranes can applied to thebase amnion layer to form the laminated tissue graft. In someembodiment, a layer of Wharton's jelly can be applied between two layersof amnion membranes, between two layers of chorion membranes, or betweenone layer of amnion membrane and one layer of chorion membrane.

In one aspect, the tissue is dehydrated by chemical dehydration followedby freeze-drying. In one aspect, the chemical dehydration step isperformed by contacting the placental tissue with a polar organicsolvent for a sufficient time and amount in order to substantially(i.e., greater than 90%, greater than 95%, or greater than 99%) orcompletely remove residual water present in the placental tissue (i.e.,dehydrate the tissue). The solvent can be protic or aprotic. Examples ofpolar organic solvents useful herein include, but are not limited to,alcohols, ketones, ethers, aldehydes, or any combination thereof.Specific, non-limiting examples include DMSO, acetone, tetrahydrofuran,ethanol, isopropanol, or any combination thereof. In one aspect, theplacental tissue is contacted with a polar organic solvent at roomtemperature. No additional steps are required, and the placental tissuecan be freeze-dried directly as discussed below. In one aspect, thecondition for chemical dehydration does not induce cell lysis.

After chemical dehydration, the placental tissue is freeze-dried inorder to remove any residual water and polar organic solvent. Afterdrying, the placental tissue graft is prepared.

In one aspect, the placental tissue is placed in a freeze-dryer, and theplacental tissue is lyophilized at between −50° C. to −80° C. In anotheraspect, the placental tissue is placed in a freeze-dryer such that it ishanging in the freeze-dryer. In other aspects, the placental tissue isplaced on a substrate that can facilitate free-drying. Examples of suchsubstrates include, but are not limited to, a pan, bowl, screen, or aframe. In one aspect, one or more placental tissues can optionally belaid on a suitable drying fixture prior to freeze-drying. For example,at least two layers of hydrated chorion, at least two layers of hydratedamnion, or at least one layer of hydrated chorion and hydrated amnioncan be applied to the drying fixture. In other aspects, the placentaltissue composed of amnion membrane, Wharton's jelly, and chorion thathas not been separated can be laid on top of the drying fixture, whereone or more additional placental tissues such as amnion membrane and/orchorion can optionally be applied on top of the tissue.

The drying fixture is preferably sized to be large enough to receive theplacental tissue, fully, in laid out, flat fashion. In one aspect, thedrying fixture is made of Teflon or of Delrin, which is the brand namefor an acetal resin engineering plastic invented and sold by DuPont andwhich is also available commercially from Werner Machine, Inc. inMarietta, Ga. Any other suitable material that is heat and cutresistant, capable of being formed into an appropriate shape to receivewet tissue can also be used for the drying fixture.

In one aspect, the receiving surface of the drying fixture can havegrooves that define the product spaces, which are the desired outercontours of the tissue after it is cut and of a size and shape that isdesired for the applicable surgical procedure in which the tissue graftwill be used. For example, the drying fixture can be laid out so thatthe grooves are in a grid arrangement. The grids on a single dryingfixture may be the same uniform size or may include multiple sizes thatare designed for different surgical applications. Nevertheless, any sizeand shape arrangement can be used for the drying fixture, as will beappreciated by those skilled in the art. In another aspect, instead ofhaving grooves to define the product spaces, the drying fixture hasraised ridges or blades.

In certain aspects, the drying fixture can include a slightly raised orindented texture in the form of text, logo, name, or similar design.This textured text, logo, name, or design can be customized or privatelabeled depending upon the company that will be selling the graft ordepending upon the desired attributes requested by the end user (e.g.,surgeon). When dried, the prepared tissue graft will mold itself aroundthe raised texture or into the indented texture, essentially providing alabel within the tissue graft itself. Preferably, the texture/label canbe read or viewed on the placental tissue graft in only one orientationso that, after dehydration, an end user (e.g., a surgeon) of the driedtissue graft will be able to identify the top and bottom of theplacental tissue. In other aspects, a stamp can be imprinted on theplacental tissue graft after freeze-drying in order to differentiate thesides of the graft.

Once the placental tissue(s) is placed on the drying fixture, the dryingfixture is placed in the freeze-dryer. The use of the freeze-dryer todehydrate the placental tissue can be more efficient and thoroughcompared to other techniques such as thermal dehydration. In general, itis desirable to avoid ice crystal formation in the placental tissue asthis may damage the extracellular matrix in the tissue graft. Bychemically dehydrating the placental tissue prior to freeze-drying, thisproblem can be avoided. The chemical-dehydration and freeze-dryingmethod also minimize formation of porous structures within the tissuegraft that more likely result from conventional dehydration methods,such as direct lyophilization, and may compromise structure strength ofthe tissue graft.

The chemical-dehydration and freeze-drying method can prepare ultra drytissue graft when needed. In one aspect, the ultra-dry material canabsorb water or an aqueous solution, such as a solution comprising abiological material useful for promote wound healing, more readily whenneeded.

In another aspect, the chemical-dehydration and freeze-drying method iscapable of forming a physical barrier to prevent adhesion during woundhealing as compared with conventional dehydration methods, such asdirect lyophilization.

Cutting & Packaging (Step 150)

Once the tissue graft has been adequately dehydrated, the tissue graftis then ready to be cut into specific product sizes and appropriatelypackages for storage, terminal sterilization, and later surgical use.The number of grafts to be produced is estimated based on the size andshape of the tissue on the drying fixture(s). An appropriate number ofpouches, one for each tissue graft, are then also introduced into thesterile/controlled environment.

If the drying fixture has grooves, then the following exemplaryprocedure is followed for cutting the tissue graft into product sizes.If the drying fixture is configured in a grid pattern, a #20 or similarstraight or rolling blade is used to cut along each groove line inparallel. Then, all lines in the perpendicular direction are cut.Alternatively, if the drying fixture has raised edges or blades, thenthe following procedure is followed for cutting the tissue graft intoproduct sizes. A sterile roller is used to roll across the dryingfixture. Sufficient pressure must be applied so that the dehydratedtissue graft is cut along all of the raised blades or edges of thedrying fixture.

After cutting, each tissue graft is placed in a respective “inner”pouch. The inner pouch, which preferably has a clear side and an opaqueside, should be oriented clear side facing up. The tissue graft isplaced in the “inner” pouch so that the texture in the form of text,logo, name, or similar design is facing out through the clear side ofthe inner pouch and is visible outside of the inner pouch. This processis repeated for each separate graft.

Each tissue graft is then given a final inspection to confirm that thereare no tears or holes, that the product size (as cut) is withinapproximately 1 millimeter (plus or minus) of the specified size forthat particular graft, that there are no noticeable blemishes ordiscoloration of the tissue, and that the textured logo or wording isreadable and viewable through the “inner” pouch. The final tissue graftscan be stored at room temperature for extended periods of time.

II. Applications of Tissue Grafts

In one aspect, provided is a wound dressing comprising the tissue graftdescribed herein.

In another aspect, provided is a method for enhancing wound healing in asubject comprising applying the tissue graft described herein to thewound.

In another aspect, provided is a method for preventing or reducing scarformation on or near the spine of a subject after a surgical procedure,the method comprising applying to the subject a tissue graft describedherein directly to the spinal dura of the subject or a region near thespine. In some embodiments, the surgical procedure comprises a posteriorprocedure. In some embodiments, the posterior procedure is a laminectomyor discectomy. In some embodiments, the posterior procedure is anAnterior Lumbar Interbody Fusion (ALIF) and Transforaminal InterbodyFusion (TLIF). In some embodiments, the surgical procedure comprises ananterior procedure. In some embodiments, the method comprises applyingthe tissue graft directly to the dural tear.

In another aspect, provided is a use of the tissue graft describedherein in cranial dura repair, a perioplastic procedure, in theelimination of a frenum pull, the regeneration of lost patella tissue,the repair of the Schneiderian membrane in the sinus cavity, soft tissuearound dental implants, vestibuloplasty, and guided tissue regeneration.

In some embodiments, the use is in a dental application. In someembodiments, the use is in an orthopedic application. In someembodiments, the use is in an ENT application. In some embodiments, theuse is in an opthalmological application (e.g., on-lay grafts ocularsurface repair). In some embodiments, the use is in a gynecologicalapplication. In some embodiments, the use is in an urologicalapplication. In some embodiments, the use is in a general surgery. Insome embodiments, the use is in a cardiac application. In someembodiments, the use is to reduce or prevent scar formation afterplastic surgery.

The grafts described herein can be used in numerous medical applicationsinvolving wound healing in a subject. In one aspect, the graftsdescribed herein are useful in enhancing or improving wound healing. Thetypes of wounds that present themselves to physicians on a daily basesare diverse. Acute wounds are caused by surgical intervention, traumaand burns. Chronic wounds are wounds that are delayed in closingcompared to healing in an otherwise healthy individual. Examples ofchronic wound types plaguing patients include diabetic foot ulcers,venous leg ulcers, pressure ulcers, arterial ulcers, and surgical woundsthat become infected.

The physician's goal when treating traumatic wounds is to heal the woundwhile allowing the patient to retain natural function in the area of thewound with minimal scaring and infection. If a wound becomes infected,it can lead to a loss of limb or life. For the most part, physiciansheal these patients without incident. However, physicians dealing withchronic wounds are mainly concerned with closing the wound as quickly aspossible to minimize the risk of an infection that could lead to loss oflimb or life. Chronic wounds are wounds on patients that havecomorbidities that complicate or delay the healing cascade. In oneaspect, the grafts described herein can function as a tissueregeneration template that delivers essential wound healing factors,extracellular matrix proteins and inflammatory mediators to help reduceinflammation, enhance healing, and reduce scar tissue formation.

In another aspect, the tissue grafts described herein are useful foraddressing or alleviating complications to the spine and surroundingregions that occur after surgery. Acute and chronic spinal injuries andpain can be attributed to trauma and/or degenerative changes in thespinal column. For the degenerative patient, there is usually aprogression of possible surgeries depending on the patient's symptomsand disease state. The first surgical option when conservative therapyhas failed is a laminectomy or micro-discectomy. These minimallyinvasive procedures are intended to relieve the pain generator orstenosis of the spinal canal. If there is progression of the disease,then other surgeries may be necessary including, but not limited to, aspinal fusion. Spinal fusions may be achieved through severalapproaches: anterior (from the front through the abdomen), posterior(from the back), or lateral (through the side). Each approach hasadvantages and disadvantages. The goal is typically to remove the spinaldisc, restore disc height and fuse the two spinal vertebrae together tolimit motion and further degradation. There are also surgical optionsfor the surgeon and patient to replace the spinal disc with anartificial disc. Spine trauma is typically treated by fusing the spinelevels or if a vertebrae is crushed, the surgeon may choose to do acorpectomy and fusing across the levels that were affected.

In one aspect, the tissue grafts described herein are useful inpreventing or reducing scar formation on the spine or near the spine andsealing dural tears. Scar formation at or near the spine after surgerycan be very debilitating and possibly require subsequent operations toaddress the symptoms as discussed above. The term “anti-adhesion” isalso used in the art to refer to the prevention of scar tissue at ornear the spine. In other aspects, the tissue grafts described herein canbe used as a protective barrier, where the graft protects the spinaldura from post-surgical trauma from the surrounding surgical site. Forexample, the grafts can prevent damage to the spinal dura caused bysharp edges from newly cut bone such as vertebrae. In other aspects, thetissue grafts can be used for anterior lumbar interbody fusion,posterior lumbar interbody fusion trans-lumbar interbody fusion,anterior cervical discectomy and fusion, micro discectomy, spinal durarepair, and as a dura sealant to prevent CSF leakage.

Depending upon the surgical procedure, the tissue graft can be applieddirectly to the spinal dura, the surrounding region of the spine toinclude nerve roots, or a combination thereof. Due to the uniquestructure of vertebrae, the tissue graft can be cut into any shape ordimension so that it can be placed and affixed at the appropriateposition in the subject. For example, when the tissue graft is used forbi-lateral coverage, membranes in the shape of a rectangle allow thetissue graft to fit around the posterior spinal process, which minimizeslateral movement. In addition to minimizing lateral movement, the tissuegraft can also provide proximal and distal barrier coverage where thespinal lamina has been removed for exposure to the affected area. In oneaspect, to ensure proper placement, the graft can be embossed on theexposed basement membrane of the graft to ensure proper placement of thegraft in the subject. In particular, proper graft placement will ensurethat the basement membrane of the graft is in direct contact with thespinal dura or surrounding region. For example, proper membraneplacement and orientation is important when applying the material inspinal applications where a posterior or anterior approach is utilized.

The grafts are useful in preventing or reducing scar formation that canresult from a variety of surgical procedures associated with the spine.The grafts can be used after any procedure in the neck, mid-back, orlower back. Depending upon the application, the epithelium of the amnionmembrane can be substantially removed. For example, in posteriorprocedures such as a laminectomy or discectomy, the epithelium layer issubstantially removed. Removal of the epithelial cell layer exposes theamnion's basement membrane layer, which increases cell signalingcharacteristics. This up regulation response enhances cellular migrationand expression of anti-inflammatory proteins, which inhibits fibrosis.The spinal dura is typically left unprotected following posteriorprocedures. Thus, the grafts described herein provide an unmet need inthese procedures.

In other aspects, the epithelial cell layer is not removed. For example,in anterior procedures or modified anterior procedures such as AnteriorLumbar Interbody Fusion (ALIF) and Transforaminal Interbody Fusion(TLIF), the amnion epithelium layer is not removed and remains intact.In these aspects, the grafts provide additional protection to thevertebral surgical site by maintaining separation from the peritoneum,larger vessels, and abdominal musculature. The membrane serves as areduced friction anatomical barrier against adhesions and scaring. Forexample, the grafts can prevent scar tissue binding major blood vesselsto the spine. This is a common problem with post-spinal surgery, whichrequires a second surgical procedure to address this.

In another aspect, the tissue grafts are useful in dental applications.For example, the grafts can be used around dental implants or in thetreatment of advanced gingival recession defect. In another aspect, thegrafts can be used in guided tissue regeneration.

In other aspects, the grafts described herein can be used in orthopedicapplications (i.e., sports medicine). Sports medicine includes therepair and reconstruction of various soft-tissue injuries in or aroundjoints caused by traumas, or chronic conditions brought about byrepeated motion, in active individuals and athletes. For example, sportsmedicine includes the treatment of a variety of different injuriesassociated with, but not limited to, shoulders, elbows, feet, ankleshand and wrists.

The main types of injuries include tendon and ligament sprains andruptures in the various joints, with the most common being ACL in theknee and rotator cuff in the shoulder. Non-tendon and ligamentprocedures include repair of torn knee meniscus and repair of kneecartilage which if left un-treated can lead to osteoarthritis of thejoint. Non-surgical options also include injections of anti-inflammatorydrugs to inflamed tendons (such as “tennis elbow”), injection oflubricants into joints (such as hyaluronic acid into the knee), as wellas physiotherapy and bracing.

In one aspect, the tissue grafts described herein can be used to wraptendon repairs to prevent scar formation on the healing tendon. They canalso provide a protective, enclosed environment for the repair toprogress successfully. The tissue grafts can be used as an off-the-shelftendon and ligament to replace the need to purchase an allograft orperform tendon or ligament transfer.

In other aspects, the tissue grafts described herein can be used in thereinforcement of rotator cuffs. Some rotator cuff tears are large enoughthat they require a reinforcement matrix to support the repair due tolack of viable native tissue. The tissue grafts described herein can beused as a matrix to reinforce a repair. In one aspect, the tissue graftsdescribed herein can be used to repair knee cartilage. For example, thetissue grafts can be used as a barrier to hold cell culturedchondrocytes or other pro-cartilage regeneration matrix inside achondral defect. In this aspect, the tissue graft would be utilized as aflap to close the defect and hold the matrix in place.

In one aspect, the tissue grafts can be used to repair peripheralnerves. The tissue graft can be used as a wrap on nerve repairs toprevent scar formation onto the healing nerve. The tissue graft can alsoprovide a protective enclosed environment for the repair to progresssuccessfully. In other aspects, the tissue grafts can be manufacturedinto a nerve regeneration tube to guide nerve growth in a protectiveenvironment where the nerve ends cannot be re-approximated. Here, nervescan re-attach up to a certain distance if the ends are allowed to meetfreely without other soft tissue interfering. In another aspect, thetissue graft can be used to wrap nerve bundles after prostatectomyprocedures. These nerves are responsible for erectile function andpossible continence. The tissue grafts can be laid on the nerves to keepthem from scarring and possibly damaging the nerves.

In other aspects, the tissue grafts described herein can be used inother orthopedic applications such as aid in the repair of periostium;help repair ruptured/damaged bursa; help secure void filling materialduring bone repair; or in applications involving a subject's extremities(e.g., anti-adhesion barrier for small bone fixation, anti-adhesionbarrier where metal plating or hardware is used, or help repairruptured/damaged bursa).

In another aspect, the tissue grafts can be used in obstetrics andgynecological (OB/GYN) surgical procedures involving the treatment ofdiseases that may be related to the fertility of the female, pain causedby the reproductive system or cancer in the reproductive system. Theseprocedures include the removal of uterine fibroids (myomectomy), removalof ovarian cysts, tubal ligations, endometriosis treatments, removal ofsome cancerous or non-cancerous tumors, and vaginal slings. Theseprocedures may be completed through a transvaginal, abdominal orlaproscopical approach.

The tissue grafts can be used as a patch to reduce the amount of scartissue in the reproductive system after a surgical procedure. Scartissue is another form of fibrous tissue and may also contribute tofertility problems. The ability to minimize the amount of scar on theovaries, or within the fallopian tubes may help with post-operativefertility and even pain. In another aspect, the tissue grafts can beused to reline the uterine wall after severe endometriosis treatmentsand increase the patient's ability to conceive. In a further aspect, thetissue graft can be used as an anti-adhesion barrier after removal ofovarian cyst or aid in the repair of vaginal wall erosion.

In other aspects, the tissue grafts can be used in cardiac applications.Angina is severe chest pain due to ischemia (a lack of blood, thus alack of oxygen supply) of the heart muscle, generally due to obstructionor spasm of the coronary arteries (the heart's blood vessels). Coronaryartery disease, the main cause of angina, is due to atherosclerosis ofthe cardiac arteries. Various open cardiac and vascular surgeryprocedures to remove atherosclerotic clots require the repair,reconstruction and closure of the vessel, and the support of aregenerative tissue patch to close and patch the surgical defect. Heartby-pass grafts and heart defect reconstruction (as part of an open-heartsurgical procedure) also can benefit from a patch or graft to provide abuttress to soft-tissue weakness, tissue replacement if there is a lackof suitable tissue, and also the potential to reduce adhesions to theheart itself. The tissue grafts described herein can be used as a patchto support the repair of vascular and cardiac defects caused byoperations and complications such as carotid artery repair, coronaryartery bypass grafting, congenital heart disease, heart valve repair,and vascular repair (i.e. peripheral vessels).

The tissue grafts described herein can be used in general surgeryprocedures. For example, general surgical procedures include proceduresrelated to the abdominal cavity. These include the intestines, stomach,colon, liver, gallbladder, appendix, bile ducts and thyroid glands.Procedures may include hernias, polypectomy, cancer removal, surgicaltreatment of Crohn's and ulcerative colitis. These procedures may bedone open or laparoscopically. In other aspects, the tissue grafts canbe used to facilitate closure of anastomosis, an anti-adhesion barrierfor anastomosis, or an anti-adhesion barrier for hernia repair.

In other aspects, the tissue grafts can be used in ENT procedures.Tympanoplasty is performed for the reconstruction of the eardrum(tympanic membrane) and/or the small bones of the middle ear. There areseveral options for treating a perforated eardrum. If the perforation isfrom recent trauma, many ear, nose and throat specialists will elect towatch and see if it heals on its own. If this does not occur or frequentre-perforation occurs in the same area, surgery may be considered.Tympanoplasty can be performed through the ear canal or through anincision behind the ear. Here, the surgeon harvests a graft from thetissues under the skin around the ear and uses it to reconstruct theeardrum. The tissue grafts described herein can be used to prevent theadditional trauma associated with harvesting the patients' own tissueand save time in surgery. In other aspects, the tissue grafts can beused as a wound covering after adenoidectomy, a wound cover aftertonsillectomy, or facilitate repair of the Sniderian membrane.

In other aspects, the tissue grafts described herein can be used inplastic surgery procedures. Scar revision is surgery to improve orreduce the appearance of scars. It also restores function and correctsskin changes (disfigurement) caused by an injury, wound, or previoussurgery. Scar tissue forms as skin heals after an injury or surgery. Theamount of scarring may be determined by the wound size, depth, andlocation; the person's age; heredity; and skin characteristics includingskin color (pigmentation). Surgery involves excision of the scar andcareful closure of the defect. In one aspect, the tissue graftsdescribed herein can be used as a patch to aid in the healing andprevention of scars; and keloid or cancer revision/removal where carefulapproximation of soft-tissue edges is not achievable and scar tissue canresult. Additionally, the anti-inflammatory properties of the tissuegraft can enhance healing as well.

In other aspects, the tissue grafts can be used in opthalmologicalapplications (e.g., on-lay grafts ocular surface repair) or urologicalapplications (e.g., facilitate closure of the vas deferens duringvasectomy reversal or facilitate closure of the vas deferens resultingfrom trauma).

Depending upon the application of the graft, the graft can be soakedwith a bioactive agent such as a solution composed of naturallyoccurring growth factors sourced from platelet concentrates, eitherusing autologous blood collection and separation products, or plateletconcentrates sourced from expired banked blood; bone marrow aspirate;stem cells derived from concentrated human placental cord blood stemcells, concentrated amniotic fluid stem cells or stem cells grown in abioreactor; or antibiotics. Here, one or more membrane layers of thetissue graft absorb the bioactive agent. Upon application of the wettissue graft with bioactive agent to the wound, the bioactive agent isdelivered to the wound over time.

Although the tissue grafts described herein can be applied directly tothe tissue of a subject, they can also be applied to a wound dressingthat can subsequently be applied to the subject. For example, the wounddressing can be gauze, a bandage or wrap, or any other suitable articlecapable of containing or affixing the tissue graft that can be applieddirectly to a subject.

Various modifications and variations can be made to the articles,compositions and methods described herein. Other aspects of thearticles, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the articles,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

What is claimed:
 1. A method for enhancing wound healing in a subjectcomprising applying to the wound a placental tissue graft produced bythe method comprising: a) laminating at least two placental tissuestogether; b) chemically dehydrating the placental tissues; and c)freeze-drying the placental tissues to produce the tissue graft, whereinthe at least two placental tissues are selected from the groupconsisting of: (1) at least two layers of chorion, (2) at least twolayers of cellularized amnion membrane, and (3) at least one layer ofchorion and at least one layer of cellularized amnion membrane.
 2. Themethod of claim 1, wherein the wound is a chronic wound.
 3. The methodof claim 1, wherein the wound is an acute wound.
 4. A method forpreventing or reducing scar formation on or near the spine of a subjectafter a surgical procedure, the method comprising applying directly tothe spinal dura of the subject or a region near the spine a placentaltissue graft produced by the method comprising: a) laminating at leasttwo placental tissues together; b) chemically dehydrating the placentaltissues; and c) freeze-drying the placental tissues to produce thetissue graft, wherein the at least two placental tissues are selectedfrom the group consisting of: (1) at least two layers of chorion, (2) atleast two layers of cellularized amnion membrane, and (3) at least onelayer of chorion and at least one layer of cellularized amnion.
 5. Themethod of claim 4, wherein the surgical procedure comprises a posteriorapproach spinal procedure.
 6. The method of claim 5, wherein theposterior approach spinal procedure is a laminectomy or discectomy. 7.The method of claim 5, wherein the posterior approach spinal procedureis an Anterior Lumbar Interbody Fusion (ALIF) or Transforaminal LumbarInterbody Fusion (TLIF).
 8. The method of claim 4, wherein the surgicalprocedure comprises an anterior approach spinal procedure.
 9. The methodof claim 4, wherein the surgical procedure comprises a lateral approachspinal procedure.
 10. A method for regenerating and/or repairing tissuein a subject, the method comprising applying to the tissue in need ofregeneration and/or repair a placental tissue graft produced by themethod comprising: a) laminating at least two placental tissuestogether; b) chemically dehydrating the placental tissues; and c)freeze-drying the placental tissues to produce the tissue graft, whereinthe at least two placental tissues are selected from the groupconsisting of: (1) at least two layers of chorion, (2) at least twolayers of cellularized amnion membrane, and (3) at least one layer ofchorion and at least one layer of cellularized amnion; and wherein themethod for regenerating and/or repairing a tissue is selected from thegroup consisting of repairing a dural tear, eliminating a frenum pull,regenerating lost patella tissue, repairing damaged Schneiderianmembrane in the sinus cavity, regenerating soft tissue around dentalimplants, performing a vestibuloplasty, repairing periosteum, repairingruptured or damaged bursa, reinforcing rotator cuffs, performing atympanoplasty, repairing ocular surfaces, facilitating closure of thevas deferens facilitating closure of anastomosis, patching vasculatureand/or cardiac defects, and promoting wound healing after plasticsurgery.
 11. The method of claim 10, wherein the placental tissue graftis applied directly to spinal dura, a surrounding region of a spineincluding nerve roots, or a combination thereof.
 12. The method of claim10, wherein the placental tissue graft is applied directly to a bone.13. The method of claim 12, wherein the bone is a vertebra.